Methods for uniform metal impregnation into a nanoporous material

ABSTRACT

The methods, systems  400  and apparatus disclosed herein concern metal  150  impregnated porous substrates  110, 210.  Certain embodiments of the invention concern methods for producing metal-coated porous silicon substrates  110, 210  that exhibit greatly improved uniformity and depth of penetration of metal  150  deposition. The increased uniformity and depth allow improved and more reproducible Raman detection of analytes. In exemplary embodiments of the invention, the methods may comprise oxidation of porous silicon  110,  immersion in a metal salt solution  130,  drying and thermal decomposition of the metal salt  140  to form a metal deposit  150.  In other exemplary embodiments of the invention, the methods may comprise microfluidic impregnation of porous silicon substrates  210  with one or more metal salt solutions  130.  Other embodiments of the invention concern apparatus and/or systems  400  for Raman detection of analytes, comprising metal-coated porous silicon substrates  110, 210  prepared by the disclosed methods.

FIELD

The present methods and apparatus relate to the field of metal 150impregnation into nanoporous materials 110, 210. More particularly,certain embodiments of the invention concern methods of producingmetal-coated porous silicon 110, 210.

BACKGROUND

The sensitive and accurate detection and/or identification of singlemolecules from biological and other samples has proven to be an elusivegoal, with widespread potential uses in medical diagnostics, pathology,toxicology, biological warfare, environmental sampling, chemicalanalysis, forensics and numerous other fields. Attempts have been madeto use Raman spectroscopy and/or surface plasmon resonance to achievethis goal. When light passes through a tangible medium, a certain amountbecomes diverted from its original direction, a phenomenon known asRaman scattering. Some of the scattered light also differs in frequencyfrom the original excitatory light, due to the absorption of light andexcitation of electrons to a higher energy state, followed by lightemission at a different wavelength. The wavelengths of the Ramanemission spectrum are characteristic of the chemical composition andstructure of the light absorbing molecules in a sample, while theintensity of light scattering is dependent on the concentration ofmolecules in the sample.

The probability of Raman interaction occurring between an excitatorylight beam and an individual molecule in a sample is very low, resultingin a low sensitivity and limited applicability of Raman analysis. It hasbeen observed that molecules near roughened silver surfaces showenhanced Raman scattering of as much as six to seven orders ofmagnitude. This surface enhanced Raman spectroscopy (SERS) effect isrelated to the phenomenon of plasmon resonance, wherein metalnanoparticles exhibit a pronounced optical resonance in response toincident electromagnetic radiation, due to the collective coupling ofconduction electrons in the metal. In essence, nanoparticles of gold,silver, copper and certain other metals can function as miniature“antenna” to enhance the localized effects of electromagnetic radiation.Molecules located in the vicinity of such particles exhibit a muchgreater sensitivity for Raman spectroscopic analysis.

Attempts have been made to exploit SERS for molecular detection andanalysis, typically by coating metal nanoparticles or fabricating roughmetal films on the surface of a substrate and then applying a sample tothe metal-coated surface. However, the number of metal particles thatcan be deposited on a planar surface is limited, producing a relativelylow enhancement factor for SERS and related Raman techniques utilizingsuch surfaces. A need exists for methods of producing SERS-activesubstrates with uniform, high densities of Raman-active metal.

Metal impregnated silicon substrates have been proposed as components ofvarious electrical devices, such as field emission electron sources andlight emitting diodes. The efficiency of such devices is limited by alack of uniformity of electrical contacts, resulting fromnon-homogeneous metal impregnation. A need exists for methods ofproducing materials with homogeneous metal impregnation for highefficiency electrical devices.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the disclosedembodiments of the invention. The embodiments of the invention may bebetter understood by reference to one or more of these drawings incombination with the detailed description of specific embodimentspresented herein.

FIG. 1 illustrates an exemplary method for producing a metal-coatedporous silicon substrate 110 comprising thermal decomposition of a metalsalt solution 130. FIG. 1A shows a porous silicon substrate 110. FIG. 1Billustrates silicon oxidation, for example by plasma oxidation, to forma layer of silicon dioxide 120. FIG. 1C shows immersion of the oxidizedporous silicon 110 in a metal salt solution 130, such as a silvernitrate solution 130. FIG. 1D illustrates removal of excess metal saltsolution 130. FIG. 1E shows drying of the solution 130 to form a thinlayer of dry metal salt 140 on the porous silicon substrate 110. FIG. 1Fillustrates thermal decomposition of the dry metal salt 140 to form auniform layer of metal 150 coating the porous silicon substrate 110.

FIG. 2 illustrates another exemplary method for producing a metal-coatedporous silicon substrate 210 comprising microfluidic impregnation.

FIG. 3 illustrates an alternative embodiment of the invention fordelivering different metal plating solutions to a porous siliconsubstrate 210.

FIG. 4 shows an exemplary system 400 for detecting various targetmolecules using a metal-coated porous silicon substrate 210 and Ramandetection.

FIG. 5 illustrates the uniform deposition of an exemplary metal 150(silver) on a porous silicon substrate 110 using a thermal decompositionmethod.

FIG. 6 shows the surface-enhanced Raman spectrum for an exemplaryanalyte, rhodamine 6G (R6G) dye molecules, obtained with aplasma-oxidized, dip and decomposed (PODD) porous silicon substrate 110uniformly coated with silver 150. The PODD substrate 110 was prepared bythe method of FIG. 1. A solution of 114 μM (micromolar) R6G moleculeswas subjected to SERS (surface enhanced Raman spectroscopy) usingexcitation at 785 nm (nanometers). FIG. 6 shows the SERS emissionspectra obtained with PODD silver-coated substrates 110 of differentporosities. The various spectra were obtained at average porosities, inorder from the lowest trace to the highest trace, of 52%, 55%, 65%, 70%and 77%.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following detailed description contains numerous specific details inorder to provide a more thorough understanding of the disclosedembodiments of the invention. However, it will be apparent to thoseskilled in the art that the embodiments of the invention may bepracticed without these specific details. In other instances, devices,methods, procedures, and individual components that are well known inthe art have not been described in detail herein.

Definitions

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “analyte” and “target” refer to any atom,chemical, molecule, compound, composition or aggregate of interest fordetection and/or identification. Non-limiting examples of analytesinclude an amino acid, peptide, polypeptide, protein, glycoprotein,lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid,sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid,hormone, metabolite, cytokine, chemokine, receptor, neurotransmitter,antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor,drug, pharmaceutical, nutrient, prion, toxin, poison, explosive,pesticide, chemical warfare agent, biohazardous agent, radioisotope,vitamin, heterocyclic aromatic compound, carcinogen, mutagen, narcotic,amphetamine, barbiturate, hallucinogen, waste product and/orcontaminant. In certain embodiments of the invention, one or moreanalytes may be labeled with one or more Raman labels.

As used herein, the term “nanocrystalline silicon” refers to siliconthat comprises nanometer-scale silicon crystals, typically in the sizerange from 1 to 100 nanometers (nm). “Porous silicon” 110, 210 refers tosilicon that has been etched or otherwise treated to form a porousstructure 110, 210.

As used herein, “operably coupled” means that there is a functionalinteraction between two or more units of an apparatus and/or system. Forexample, a Raman detector 410 may be “operably coupled” to a computer ifthe computer can obtain, process, store and/or transmit data on Ramansignals detected by the detector 410.

Porous Substrates

Certain embodiments of the invention concern methods for coating poroussubstrates 110, 210 with a uniform layer of one or more metals 150, suchas Raman active metals 150. Although in particular embodiments of theinvention the porous substrates 110, 210 disclosed herein are poroussilicon substrates 110, 210, those embodiments are not limiting. Anyporous substrate 110, 210 that is resistant to the application of heatmay be used in the disclosed methods, systems 400 and/or apparatus. Incertain embodiments, application of heat to about 300° C., 400° C., 500°C., 600° C., 700° C., 800° C., 900° C. or 1,000° C. is contemplated. Insome embodiments of the invention, the porous substrate 110, 210 may berigid. A variety of porous substrates 110, 210 are known, including butnot limited to porous silicon, porous polysilicon, porous metal gridsand porous aluminum. Exemplary methods of making porous substrates 110,210 are disclosed in further detail below.

Porous polysilicon substrates 110, 210 may be made by known techniques(e.g., U.S. Pat. Nos. 6,249,080 and 6,478,974). For example, a layer ofporous polysilicon 110, 210 may be formed on top of a semiconductorsubstrate by the use of low pressure chemical vapor deposition (LPCVD).The LPCVD conditions may include, for example, a pressure of about 20pascal, a temperature of about 640° C. and a silane gas flow of about600 sccm (standard cubic centimeters) (U.S. Pat. No. 6,249,080). Apolysilicon layer may be etched, for example using electrochemicalanodization with HF (hydrofluoric acid) or chemical etching with nitricacid and hydrofluoric acid, to make it porous (U.S. Pat. No. 6,478,974).Typically, porous polysilicon 110, 210 layers formed by such techniquesare limited in thickness to about 1 μm (micrometer) or less. Incontrast, porous silicon 110, 210 can be etched throughout the thicknessof the bulk silicon wafer, which has a typical thickness of about 500μm.

Porous aluminum substrates 110, 210 may also be made by known techniques(e.g., Cai et al., Nanotechnology 13:627, 2002; Varghese et al., J.Mater. Res. 17:1162-1171, 2002). For example, nanoporous aluminum oxidethin films 110, 210 may be fabricated on silicon or silicon dioxide 120using an electrochemical-assisted self-assembly process (Cai et al.,2002). The porous aluminum film 110, 210 may be thermally annealed toimprove its uniformity (Cai et al., 2002). Alternatively, a thin layerof solid aluminum may be electrochemically anodized in dilute solutionsof oxalic acid and/or sulfuric acid to create a nanoporous alumina film110, 210 (Varghese et al., 2002). The examples disclosed herein are notlimiting and any known type of heat resistant porous substrate 110, 210may be used. Such porous substrates 110, 210 may be uniformlyimpregnated with one or more metals 150, such as silver, using themethods disclosed herein.

Nanocrystalline Porous Silicon

Nanocrystalline Silicon

Certain embodiments of the invention concern systems 400 and/orapparatus comprising one or more layers of nanocrystalline silicon.Various methods for producing nanocrystalline silicon are known (e.g.,Petrova-Koch et al., “Rapid-thermal-oxidized porous silicon—the superiorphotoluminescent Si,” Appl. Phys. Lett. 61:943, 1992; Edelberg, et al.,“Visible luminescence from nanocrystalline silicon films produced byplasma enhanced chemical vapor deposition,” Appl. Phys. Lett.,68:1415-1417, 1996; Schoenfeld, et al., “Formation of Si quantum dots innanocrystalline silicon,” Proc. 7th Int. Conf. on ModulatedSemiconductor Structures, Madrid, pp. 605-608, 1995; Zhao, et al.,“Nanocrystalline Si: a material constructed by Si quantum dots,” 1stInt. Conf. on Low Dimensional Structures and Devices, Singapore, pp.467-471, 1995; Lutzen et al., Structural characteristics of ultrathinnanocrystalline silicon films formed by annealing amorphous silicon, J.Vac. Sci. Technology B 16:2802-05, 1998; U.S. Pat. Nos. 5,770,022;5,994,164; 6,268,041; 6,294,442; 6,300,193). The methods, systems 400and apparatus disclosed herein are not limited by the method ofproducing nanocrystalline silicon and any known method may be used.

Non-limiting exemplary methods for producing nanocrystalline siliconinclude silicon (Si) implantation into a silicon rich oxide andannealing; solid phase crystallization with metal nucleation catalysts;chemical vapor deposition; PECVD (plasma enhanced chemical vapordeposition); gas evaporation; gas phase pyrolysis; gas phasephotopyrolysis; electrochemical etching; plasma decomposition of silanesand polysilanes; high pressure liquid phase reduction-oxidationreactions; rapid annealing of amorphous silicon layers; depositing anamorphous silicon layer using LPCVD (low pressure chemical vapordeposition) followed by RTA (rapid thermal anneal) cycles; plasmaelectric arc deposition using a silicon anode and laser ablation ofsilicon (U.S. Pat. Nos. 5,770,022; 5,994,164; 6,268,041; 6,294,442;6,300,193). Depending on the process, Si crystals of anywhere from 1 to100 nm or more in size may be formed as a thin layer on a chip, aseparate layer and/or as aggregated crystals. In certain embodiments ofthe invention, a thin layer comprising nanocrystalline silicon attachedto a substrate layer may be used.

In various embodiments of the invention, it is contemplated thatnanocrystalline silicon may be used to form a porous silicon substrate110, 210. However, the embodiments are not limited to as to thecomposition of the starting material, and in alternative embodiments ofthe invention it is contemplated that other materials may be utilized,provided that the material is capable of forming a porous substrate 110,210 that can be coated with a metal 150, as exemplified in FIG. 1.

In certain embodiments of the invention, the size and/or shape ofsilicon crystals and/or pore size in porous silicon 110, 210 may beselected to be within predetermined limits, for example, in order tooptimize the plasmon resonant frequency of metal-coated porous silicon110, 210 (see, e.g., U.S. Pat. No. 6,344,272). Techniques forcontrolling the size of nanoscale silicon crystals are known (e.g., U.S.Pat. Nos. 5,994,164 and 6,294,442). The plasmon resonant frequency mayalso be adjusted by controlling the thickness and/or composition of themetal layer 150 coating the porous silicon 110, 210 (U.S. Pat. No.6,344,272).

Porous Silicon

Certain embodiments of the invention concern systems 400 and/orapparatus comprising a metal-coated porous substrate 110, 210. Invarious embodiments, the substrate may comprise nanocrystalline poroussilicon 110, 210. The substrate is not limited to pure silicon, but mayalso comprise silicon nitride, silicon oxide, silicon dioxide 120,germanium and/or other materials known for chip manufacture. Other minoramounts of material may also be present, such as dopants. Porous silicon110, 210 has a large surface area of up to 783 m²/cm³, providing a verylarge surface for applications such as surface enhanced Ramanspectroscopy techniques.

Porous silicon 110, 210 was discovered in the late 1950's byelectropolishing silicon in dilute hydrofluoric acid solutions. As isknown in the art, porous silicon 110, 210 may be produced by etching asilicon substrate with dilute hydrofluoric acid (HF) in anelectrochemical cell. In certain cases, silicon may be initially etchedin HF at low current densities. After the initial pores are formed, thesilicon may be removed from the electrochemical cell and etched in verydilute HF to widen the pores formed in the electrochemical cell. Thecomposition of the porous silicon substrate 110, 210 will also affectpore size, depending on whether or not the silicon is doped, the type ofdopant and the degree of doping. The effect of doping on silicon poresize is known in the art. For embodiments of the invention involvingdetection and/or identification of large biomolecules, a pore size ofabout 2 nm to 100 or 200 nm may be selected. The orientation of pores inporous silicon 110, 210 may also be selected in particular embodimentsof the invention. For example, an etched 1,0,0 crystal structure willhave pores oriented perpendicular to the crystals, while 1,1,1 or 1,1,0crystal structures will have pores oriented diagonally along the crystalaxis. The effect of crystal structure on pore orientation is also knownin the art. Crystal composition and porosity may also be regulated tochange the optical properties of the porous silicon 110, 210. Suchproperties may be changed, for example, to enhance Raman signals anddecrease background noise and/or to optimize the characteristics oflight emitting diodes or field emission electron sources incorporatingmetal-coated porous silicon 110, 210. The optical properties of poroussilicon 110, 210 are known in the art (e.g., Cullis et al., J. Appl.Phys. 82:909-965, 1997; Collins et al., Physics Today 50:24-31, 1997).

In a non-limiting example of a method for producing a porous siliconsubstrate 110, 210, a silicon wafer may be placed inside anelectrochemical cell comprising an inert material, such as Teflon®. Thewafer is connected to the positive pole of a constant current source,forming the anode of the electrochemical cell. The negative pole of theconstant current source is connected to a cathode, such as a platinumelectrode. The electrochemical cell may be filled with a diluteelectrolyte solution of HF in ethanol. Alternatively, HF may bedissolved in other alcohols and/or surfactants known in the art, such aspentane or hexane. In certain embodiments of the invention, a computermay be operably coupled to a constant current source to regulate thecurrent, voltage and/or time of electrochemical etching. The siliconwafer exposed to HF electrolyte in the electrochemical cell becomesetched to form a porous silicon substrate 110, 210. As is known in theart, the thickness of the porous silicon layer 110, 210 and the degreeof porosity of the silicon may be controlled by regulating the timeand/or current density of anodization and the concentration of HF in theelectrolyte solution (e.g., U.S. Pat. No. 6,358,815).

In various embodiments of the invention, portions of the silicon wafermay be protected from HF etching by coating with any known resistcompound, such as polymethyl-methacrylate. Lithography methods, such asphotolithography, of use for exposing selected portions of a siliconwafer to HF etching are well known in the art. Selective etching may beof use to control the size and shape of a porous Si chamber 110, 210 tobe used for Raman spectroscopy or for various electrical devices. Incertain embodiments of the invention, a porous silicon chamber 110, 210of about 1 μm (micrometer) in diameter may be used. In other embodimentsof the invention, a trench or channel of porous silicon 110, 210 ofabout 1 μm in width may be used. The size of the porous silicon chamber110, 210 is not limiting, and it is contemplated that any size or shapeof porous silicon chamber 110, 210 may be used. A 1 μm chamber size maybe of use, for example, with an excitatory laser 410 that emits a lightbeam of about 1 μm in size.

The exemplary method above is not limiting for producing porous siliconsubstrates 110, 210 and it is contemplated that any method known in theart may be used. Non-limiting examples of methods for making poroussilicon substrates 110, 210 include anodic etching of silicon wafers anddepositing a silicon/oxygen containing material followed by controlledannealing (e.g., Canham, “Silicon quantum wire array fabrication byelectrochemical and chemical dissolution of wafers,” Appl. Phys. Lett.57:1046, 1990; U.S. Pat. Nos. 5,561,304; 6,153,489; 6,171,945;6,322,895; 6,358,613; 6,358,815; 6,359,276). In various embodiments ofthe invention, the porous silicon layer 110, 210 may be attached to oneor more supporting layers, such as bulk silicon, quartz, glass and/orplastic. In certain embodiments, an etch stop layer, such as siliconnitride, may be used to control the depth of etching. The porous siliconlayer 110, 210 may be incorporated into a semiconductor chip, usingknown methods of chip manufacture. In certain embodiments of theinvention, a metal-coated porous silicon 110, 210 chamber may bedesigned as part of an integral chip, connected to various channels,microchannels, nanochannels, microfluidic channels, reaction chambers,solvent reservoirs 220, waste reservoirs 230, etc. In alternativeembodiments, a metal-coated porous silicon 110, 210 chamber may be cutout of a silicon wafer and incorporated into a chip and/or other device.

In certain alternative embodiments of the invention, it is contemplatedthat additional modifications to the porous silicon substrate 110, 210may be made, either before or after metal 150 coating. For example,after etching a porous silicon substrate 110, 210 may be oxidized, usingmethods known in the art, to silicon oxide and/or silicon dioxide 120.Oxidation may be used, for example, to increase the mechanical strengthand stability of the porous silicon substrate 110, 210 and/or to preventspontaneous immersion plating of porous silicon 110, 210, which can leadto pore blockage of nanoscale channels. Alternatively, the metal-coatedporous silicon substrate 110, 210 may be subjected to further etching toremove the silicon material, leaving a metal 150 shell that may be lefthollow or may be filled with other materials, such as one or moreadditional metals 150.

Metal Coating of Porous Substrates

Porous substrates 110, 210, such as porous silicon 110, 210, may becoated with a metal 150, such as a Raman active metal 150. ExemplaryRaman active metals 150 include, but are not limited to gold, silver,platinum, copper and aluminum. Known methods of metal 150 coatinginclude electroplating; cathodic electromigration; evaporation andsputtering of metals 150; using seed crystals to catalyze plating (i.e.using a copper/nickel seed to plate gold); ion implantation; diffusion;or any other method known in the art for plating thin metal layers 150on porous substrates 110, 210. (See, e.g., Lopez and Fauchet, “Erbiumemission from porous silicon one-dimensional photonic band gapstructures,” Appl. Phys. Lett. 77:3704-6, 2000; U.S. Pat. Nos.5,561,304; 6,171,945; 6,359,276.) Another non-limiting example of metal150 coating comprises electroless plating (e.g., Gole et al., “Patternedmetallization of porous silicon from electroless solution for directelectrical contact,” J. Electrochem. Soc. 147:3785, 2000). Thecomposition and/or thickness of the metal layer 150 may be controlled tooptimize optical and/or electrical characteristics of the metal-coatedporous substrates 110, 210.

Arsenic-anodized porous silicon 110, 210 is known to function as amoderate reducing agent for metal ions, thereby initiating spontaneousimmersion plating of metal 150 on the top surface of the porous area110, 210 and closing the pore openings. Thus, using standard methods ofmetal 150 impregnation, it is difficult to obtain a uniform metal 150depth profile while maintaining an open porous surface 110, 210. Thereis a trade-off between the unblocked pores and metal 150 penetrationdepth, which can be explained as follows. High concentrations of metalion are needed to obtain a better metal 150 depth profile. However,exposure to high concentrations of metal salt solutions 130 close thepores due to the thick metal film 150 deposition from the spontaneousimmersion plating reaction. To maintain an open pore, the concentrationof metal ion in solution 130 needs to be lower. However, this causespoorer penetration depth, as well as reducing the amount of metal 150deposited. This problem is resolved by the methods disclosed herein,which allow a more uniform metal 150 deposition without pore clogging.

Metal Coating by Thermal Decomposition of a Metal Salt

As illustrated in FIG. 1, in particular embodiments of the invention aporous silicon substrate 110 may be uniformly coated with a metal 150,such as a Raman sensitive metal 150, by a method comprising thermaldecomposition of a metal salt layer 140. In particular embodiments ofthe invention, the metal 150 is silver. A porous silicon substrate 110(FIG. 1A) may be obtained, for example, as disclosed above. To preventpremature metal 150 deposition and pore blocking, the surface layer ofsilicon may be oxidized to silicon dioxide 120 (FIG. 1B), for example bychemical oxidation or plasma oxidation. Oxidation prevents spontaneousimmersion plating by stabilizing the porous silicon 110 surface. In theabsence of oxidation, positively charged silver cations can engage in aredox reaction with unoxidized silicon, resulting in spontaneous silvermetal 150 deposition.

Following oxidation, the porous silicon substrate 110 is wet with ametal salt solution 130, such as a 1 M solution of silver nitrate(AgNO₃) (FIG. 1C). In a non-limiting example, the oxidized poroussilicon substrate 110 is dipped into a silver nitrate solution 130 for20 minutes, until the pores are completely wet with the silver nitratesolution 130. Excess metal salt solution 130 is removed, for example, bynitrogen gun drying (FIG. 1D). The solution 130 remaining in the poresmay be dried, for example, by heating to 100° C. for 20 min. At thispoint, the solvent has evaporated and a thin layer of dry silver nitratesalt 140 is deposited on the surface of the porous silicon 110. The drysalt 140 may be thermally decomposed (FIG. 1F), for example by heatingto 500° C. for 30 min in an ambient pressure furnace. The reaction ofEquation 1 occurs spontaneously at temperatures above 573° K. (about300° C.). The nitrate ion is converted to gaseous nitrogen dioxideaccording to Equation 1, resulting in deposition of a uniform layer ofmetallic silver 150 coating the porous silicon substrate 110 (FIG. 1F).Although nitrogen dioxide has been used as a photoetching agent, underthe conditions of the disclosed method it does not appear to etch thesilicon dioxide layer 120.AgNO₃→Ag(s)+NO₂(gas)+½O₂(gas)  (1)

The thickness of the deposited metal layer 150 may be controlled, forexample, by varying the concentration of the metal salt solution 130.Depending on the thickness of metal layer 150 to be deposited, the saltsolution 130 concentration can vary between a wide range, of about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5,3.0, 3.5, 4.0, 4.5 to 5.0 M (molar). Although the exemplary methodutilizes a silver solution 130, the embodiments of the invention are notlimited to depositing silver 150 but may encompass any known metal 150,including but not limited to Raman active metals 150 such as gold,copper, platinum, aluminum, etc. The methods are also not limited as tothe type of salt used. In certain embodiments of the invention, theanionic species used to form the metal salt may be one that is convertedto a gaseous species and driven off during the thermal decompositionprocess, such as nitrate or sulfate ion. However, in alternativeembodiments any anionic species without limitation may be used.

Metal Coating by Microfluidic Impregnation

In alternative embodiments of the invention illustrated in FIG. 2, aporous membrane 210, such as a porous silicon membrane 210, may becoated with metal 150 using microfluidic impregnation. In an exemplarymethod, a porous silicon membrane 210 may be obtained as disclosedabove. The porous silicon layer 210 may be electropolished and suspendedin a solution. The electropolished membrane 210 may be inserted into amicrofluidic pathway between one or more solvent reservoirs 220 and awaste reservoir 230 that are connected through cross-paths 240. Suchmicrofluidic pathways may be produced by any method known in the art,such as micromolding with PDMS (polydimethyl siloxane), standardlithography techniques or photolithography and etching of various chipmaterials (e.g., Duffy et al., Anal. Chem. 70:4974-84, 1998). The poroussilicon membrane 210 may be incorporated into any type of microfluidicsystem. In certain embodiments of the invention, microfluidic systemsincorporating porous silicon membranes 210 may be of use for a widevariety of applications relating to analysis and/or separation ofpolymer molecules, including but not limited to proteins and nucleicacids. Methods for micro and/or nanoscale manufacturing are known in theart, as discussed in more detail below.

A metal salt solution 130, such as a silver nitrate solution 130, may beintroduced through the solvent reservoir 220 and allowed to flow throughthe porous silicon membrane 210 to a waste reservoir 230. A spontaneousreaction will occur, as indicated in Equation 2.Ag⁺(aq.)+Si(surface)+2H₂O(liquid)→Ag(solid)+H₂(gas)+SiO₂(surface)+2H⁺  (2)

As disclosed in Equation 2, an aqueous metal solution 130 reactsspontaneously with a porous silicon surface 210 in a redox reaction,producing a deposited metal 150 coating on the porous silicon 210. Thethickness of the metal 150 coating may be controlled by the metal saltconcentration of the solution 130, the rate of flow through themicrofluidic pathway, the temperature, and/or the length of time thatthe solution 130 is allowed to flow through the membrane 210. Techniquesfor controlling such metal 150 plating reactions are known in the art.

The method is not limited to silver solutions 130, but may also beperformed with solutions 130 of other metal salts, including but notlimited to Raman active metals 150 such as gold, platinum, aluminum,copper, etc. In other alternative embodiments of the invention, theporous silicon membrane 210 may be coated with two or more differentmetals 150, using multiple solvent reservoirs 220 containing differentmetal plating solutions 130 (FIG. 3). In certain embodiments of theinvention, one or more reservoirs 220 may contain a wash solution toremove excess metal plating solution 130. Coating with multiple metals150 may be used to manipulate the electrical, optical and/or Ramansurface characteristics of the metal-coated porous silicon membrane 210,such as the degree of surface enhancement of the Raman signal, thedistance from the surface 210 at which resonance occurs, the range ofwavelengths of Raman resonance, etc.

The disclosed methods result in the production of a metal-coated poroussilicon membrane 210 integrated into a microfluidic pathway. Such anintegrated microchip may be directly incorporated into a Raman detectionsystem 400 as exemplified in FIG. 4. One or more samples suspected ofcontaining target molecules may be loaded into corresponding solventreservoirs 220. Samples may be channeled through the microfluidicpathway to enter the metal-coated membrane 210. Once in the membrane210, the target molecule may be excited by an excitatory light source410, such as a laser 410. An emitted Raman signal may be detected by aRaman detector 420, as discussed in more detail below. Once analyzed,samples may be removed into a waste reservoir 230, the membrane 210washed and the next sample analyzed. The Raman detection system 400 mayincorporate various components known in the art, such as Raman detectors420 and excitatory light sources 410, or may comprise custom componentsdesigned to be fully integrated into the system 400 to optimize Ramandetection of analytes.

Micro-Electro-Mechanical Systems (MEMS)

In some embodiments of the invention, a metal-coated porous siliconsubstrate 110, 210 may be incorporated into a larger apparatus and/orsystem 400. In certain embodiments, the substrate 110, 210 may beincorporated into a micro-electro-mechanical system (MEMS) 400. MEMS areintegrated systems 400 comprising mechanical elements, sensors,actuators, and electronics. All of those components may be manufacturedby known microfabrication techniques on a common chip, comprising asilicon-based or equivalent substrate (e.g., Voldman et al., Ann. Rev.Biomed. Eng. 1:401-425, 1999). The sensor components of MEMS may be usedto measure mechanical, thermal, biological, chemical, optical and/ormagnetic phenomena. The electronics may process the information from thesensors and control actuator components such pumps, valves, heaters,coolers, filters, etc. thereby controlling the function of the MEMS.

The electronic components of MEMS may be fabricated using integratedcircuit (IC) processes (e.g., CMOS, Bipolar, or BICMOS processes). Theymay be patterned using photolithographic and etching methods known forcomputer chip manufacture. The micromechanical components may befabricated using compatible “micromachining” processes that selectivelyetch away parts of the silicon wafer or add new structural layers toform the mechanical and/or electromechanical components.

Basic techniques in MEMS manufacture include depositing thin films ofmaterial on a substrate, applying a patterned mask on top of the filmsby photolithographic imaging or other known lithographic methods, andselectively etching the films. A thin film may have a thickness in therange of a few nanometers to 100 micrometers. Deposition techniques ofuse may include chemical procedures such as chemical vapor deposition(CVD), electrodeposition, epitaxy and thermal oxidation and physicalprocedures like physical vapor deposition (PVD) and casting. Methods formanufacture of nanoelectromechanical systems may be used for certainembodiments of the invention. (See, e.g., Craighead, Science290:1532-36, 2000.)

In some embodiments of the invention, metal-coated porous siliconsubstrates 110, 210 may be connected to various fluid filledcompartments, such as microfluidic channels, nanochannels and/ormicrochannels. These and other components of the apparatus may be formedas a single unit, for example in the form of a chip as known insemiconductor chips and/or microcapillary or microfluidic chips.Alternatively, the metal-coated porous silicon substrate 110, 210 may beremoved from a silicon wafer and attached to other components of anapparatus. Any materials known for use in such chips may be used in thedisclosed apparatus, including silicon, silicon dioxide 120, siliconnitride, polydimethyl siloxane (PDMS), polymethylmethacrylate (PMMA),plastic, glass, quartz, etc.

Techniques for batch fabrication of chips are well known in the fieldsof computer chip manufacture and/or microcapillary chip manufacture.Such chips may be manufactured by any method known in the art, such asby photolithography and etching, laser ablation, injection molding,casting, molecular beam epitaxy, dip-pen nanolithography, chemical vapordeposition (CVD) fabrication, electron beam or focused ion beamtechnology or imprinting techniques. Non-limiting examples includeconventional molding with a flowable, optically clear material such asplastic or glass; photolithography and dry etching of silicon dioxide120; electron beam lithography using polymethylmethacrylate resist topattern an aluminum mask on a silicon dioxide 120 substrate, followed byreactive ion etching. Known methods for manufacture ofnanoelectromechanical systems may be used for certain embodiments of theinvention. (See, e.g., Craighead, Science 290:1532-36, 2000.) Variousforms of microfabricated chips are commercially available from, e.g.,Caliper Technologies Inc. (Mountain View, Calif.) and ACLARA BioSciencesInc. (Mountain View, Calif.).

In certain embodiments of the invention, part or all of the apparatusmay be selected to be transparent to electromagnetic radiation at theexcitation and emission frequencies used for Raman spectroscopy, such asglass, silicon, quartz or any other optically clear material. Forfluid-filled compartments that may be exposed to various biomolecules,such as proteins, peptides, nucleic acids, nucleotides and the like, thesurfaces exposed to such molecules may be modified by coating, forexample to transform a surface from a hydrophobic to a hydrophilicsurface and/or to decrease adsorption of molecules to a surface. Surfacemodification of common chip materials such as glass, silicon, quartzand/or PDMS is known in the art (e.g., U.S. Pat. No. 6,263,286). Suchmodifications may include, but are not limited to, coating withcommercially available capillary coatings (Supelco, Bellafonte, Pa.),silanes with various functional groups such as polyethyleneoxide oracrylamide, or any other coating known in the art.

Raman Spectroscopy

In certain embodiments of the invention, the disclosed methods, systems400 and apparatus are of use for the detection and/or identification ofanalytes by surface enhanced Raman spectroscopy (SERS), surface enhancedresonance Raman spectroscopy (SERRS) and/or coherent anti-Stokes Ramanspectroscopy (CARS) detection. Compared to existing techniques, thedisclosed methods, systems 400 and apparatus provide SERS activesubstrates with increased and more uniform metal 150 density and greaterdepth of field of SERS enhancement, allowing more efficient Ramandetection and/or identification of analytes.

Previous methods for SERS detection of various analytes have usedcolloidal metal 150 particles, such as aggregated silver 150nanoparticles, that were typically coated onto a substrate and/orsupport (e.g., U.S. Pat. Nos. 5,306,403; 6,149,868; 6,174,677;6,376,177). While such arrangements occasionally allow SERS detectionwith as much as 10⁶ to 10⁸ increased sensitivity, they are not capableof single molecule detection of small analytes such as nucleotides, asdisclosed herein. Enhanced sensitivity of Raman detection is apparentlynot uniform within a colloidal particle aggregate, but rather depends onthe presence of “hot spots.” The physical structure of such hot spots,the range of distances from the metal 150 nanoparticles at whichenhanced sensitivity occurs, and the spatial relationships betweenaggregated nanoparticles and analytes that allow enhanced sensitivityhave not been characterized. Further, aggregated metal 150 nanoparticlesare inherently unstable in solution, with adverse effects on thereproducibility of single molecule detection. The present methods,systems 400 and apparatus provide a stable microenvironment for SERSdetection in which the physical conformation and density of theRaman-active metal 150 porous substrate 110, 210 may be preciselycontrolled, allowing reproducible, sensitive and accurate detection ofanalytes in solution.

Raman Detectors

In some embodiments of the invention, analytes may be detected and/oridentified by any known method of Raman spectroscopy. In suchembodiments, the metal-coated porous substrate 110, 210 may be operablycoupled to one or more Raman detection units. Various methods fordetection of analytes by Raman spectroscopy are known in the art. (See,e.g., U.S. Pat. Nos. 6,002,471; 6,040,191; 6,149,868; 6,174,677;6,313,914). Variations on surface enhanced Raman spectroscopy (SERS),surface enhanced resonance Raman spectroscopy (SERRS), hyper-Ramanspectroscopy and coherent anti-Stokes Raman spectroscopy (CARS) havebeen disclosed. In SERS and SERRS, the sensitivity of the Ramandetection is enhanced by a factor of 10⁶ or more for molecules adsorbedon roughened metal 150 surfaces, such as silver, gold, platinum, copperor aluminum surfaces.

A non-limiting example of a Raman detection unit is disclosed in U.S.Pat. No. 6,002,471. An excitation beam may be generated by either afrequency doubled Nd:YAG laser 410 at 532 nm wavelength or a frequencydoubled Ti:sapphire laser 410 at 365 nm wavelength. Alternatively,excitation beams may be generated at 785 nm using a Ti:sapphire laser410 or 514 nm using an argon laser 410. Pulsed laser beams or continuouslaser beams may be used. The excitation beam passes through confocaloptics and a microscope objective, and is focused onto the Raman activesubstrate 110, 210 containing one or more analytes. The Raman emissionlight from the analytes is collected by the microscope objective and theconfocal optics and is coupled to a monochromator for spectraldissociation. The confocal optics includes a combination of dichroicfilters, barrier filters, confocal pinholes, lenses, and mirrors forreducing the background signal. Standard full field optics can be usedas well as confocal optics. The Raman emission signal is detected by aRaman detector 420, comprising an avalanche photodiode interfaced with acomputer for counting and digitization of the signal.

Another example of a Raman detection unit is disclosed in U.S. Pat. No.5,306,403, including a Spex Model 1403 double-grating spectrophotometerwith a gallium-arsenide photomultiplier tube (RCA Model C31034 or BurleIndustries Model C3103402) operated in the single-photon counting mode.The excitation source comprises a 514.5 mn line argon-ion laser 410 fromSpectraPhysics, Model 166, and a 647.1 nm line of a krypton-ion laser410 (Innova 70, Coherent).

Alternative excitation sources include a nitrogen laser 410 (LaserScience Inc.) at 337 nm and a helium-cadmium laser 410 (Liconox) at 325nm (U.S. Pat. No. 6,174,677), a light emitting diode 410, an Nd:YLFlaser 410, and/or various ion lasers 410 and/or dye lasers 410. Theexcitation beam may be spectrally purified with a bandpass filter(CHROMA) and may be focused on the Raman active substrate 110, 210 usinga 20× objective lens (Nikon). The objective lens may be used to bothexcite the analytes and to collect the Raman signal, by using aholographic beam splitter (Kaiser Optical Systems, Inc., Model HB647-26N18) to produce a right-angle geometry for the excitation beam andthe emitted Raman signal. A holographic notch filter (Kaiser OpticalSystems, Inc.) may be used to reduce Rayleigh scattered radiation.Alternative Raman detectors 420 include an ISA HR-320 spectrographequipped with a red-enhanced intensified charge-coupled device (RE-ICCD)detection system (Princeton Instruments). Other types of detectors 420may be used, such as Fourier-transform spectrographs (based onMichaelson interferometers), charged injection devices, photodiodearrays, InGaAs detectors, electron-multiplied CCD, intensified CCDand/or phototransistor arrays.

Any suitable form or configuration of Raman spectroscopy or relatedtechniques known in the art may be used for detection of analytes,including but not limited to normal Raman scattering, resonance Ramanscattering, surface enhanced Raman scattering, surface enhancedresonance Raman scattering, coherent anti-Stokes Raman spectroscopy(CARS), stimulated Raman scattering, inverse Raman spectroscopy,stimulated gain Raman spectroscopy, hyper-Raman scattering, molecularoptical laser examiner (MOLE) or Raman microprobe or Raman microscopy orconfocal Raman microspectrometry, three-dimensional or scanning Raman,Raman saturation spectroscopy, time resolved resonance Raman, Ramandecoupling spectroscopy or UV-Raman microscopy.

Raman Labels

Certain embodiments of the invention may involve attaching a label toone or more analytes to facilitate their measurement by the Ramandetection unit. Non-limiting examples of labels that could be used forRaman spectroscopy include TRIT (tetramethyl rhodamine isothiol), NBD(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,terephthalic acid, isophthalic acid, cresyl fast violet, cresyl blueviolet, brilliant cresyl blue, para-aminobenzoic acid, erythrosine,biotin, digoxigenin, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxy rhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino phthalocyanines, azomethines, cyanines,xanthines, succinylfluoresceins, aminoacridine, quantum dots, carbonnanotubes and fullerenes. These and other Raman labels may be obtainedfrom commercial sources (e.g., Molecular Probes, Eugene, Oreg.; SigmaAldrich Chemical Co., St. Louis, Mo.) and/or synthesized by methodsknown in the art.

Polycyclic aromatic compounds may function as Raman labels, as is knownin the art. Other labels that may be of use for particular embodimentsof the invention include cyanide, thiol, chlorine, bromine, methyl,phosphorus and sulfur. The use of labels in Raman spectroscopy is known(e.g., U.S. Pat. Nos. 5,306,403 and 6,174,677). The skilled artisan willrealize that the Raman labels used should generate distinguishable Ramanspectra and may be specifically bound to or associated with differenttypes of analytes.

Labels may be attached directly to the analytes or may be attached viavarious linker compounds. Cross-linking reagents and linker compounds ofuse in the disclosed methods are known in the art. Raman labels thatcontain reactive groups designed to covalently react with othermolecules, such as analytes, are commercially available (e.g., MolecularProbes, Eugene, Oreg.). Methods for preparing labeled analytes are known(e.g., U.S. Pat. Nos. 4,962,037; 5,405,747; 6,136,543; 6,210,896).

EXAMPLES Example 1 Construction of a Porous Silicon Substrate

Formation of Porous Nanocrystalline Silicon

Methods for making nanocrystalline porous silicon 110, 210 are known inthe art (e.g., U.S. Pat. No. 6,017,773). A layer of nanocrystallineporous silicon 110, 210 may be formed electrochemically as disclosed inPetrova-Koch et al. (Appl. Phys. Let. 61:943, 1992). Depending on theparticular application, the silicon may be lightly or heavily p-doped orn-doped prior to etching to regulate the characteristics of the poroussilicon substrate 110, 210. Single crystal silicon ingots may be grownby the well known Czochralski method (e.g.,http://www.msil.ab.psiweb.com/english/msilhist4-e.html). A singlecrystal silicon wafer may be treated with anodic etching in diluteHF/electrolyte to form a nanocrystalline porous silicon substrate 110,210. Alternatively, chemical etching in a solution of HF, nitric acidand water may be used without anodic etching. Ethanol may be used as awetting agent to improve pore wetting with the HF solution.

The wafer may be coated with polymethyl-methacrylate resist or any otherknown resist compound before etching. A pattern for the nanocrystallineporous silicon substrate 110, 210 may be formed by standardphotolithographic techniques. In different embodiments of the invention,the nanocrystalline porous substrate 110, 210 may be circular, trenchshaped, channel shaped or of any other selected shape. In certainembodiments, multiple porous substrates 110, 210 may be formed on asingle silicon wafer to allow for multiple sampling channels and/orchambers for Raman analysis. Each sampling channel and/or chamber may beoperably coupled to one or more Raman detectors 420.

After resist coating and lithography, the wafer may be exposed to asolution of between about 15 to 50 weight percent HF in ethanol and/ordistilled water in an electrochemical cell comprised of Teflon®. Etchingmay be performed in the dark (p-type silicon) or in the light (n-type orp-type silicon). In different embodiments of the invention, the entireresist coated wafer may be immersed in an HF solution. In alternativeembodiments, the wafer may be held in place in the electrochemical cell,for example using a synthetic rubber washer, with only a portion of thewafer surface exposed to the HF solution (U.S. Pat. No. 6,322,895). Ineither case, the wafer may be electrically connected to the positivepole of a constant current source to form the anode of theelectrochemical cell. A platinum electrode may provide the cathode forthe cell. The wafer may be etched using an anodization current densityof between 5 to 250 milliamperes/cm² for between 5 seconds to 30 minutesin the dark, depending on the selected degree of porosity. In particularembodiments of the invention, a porosity of about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or 90% may be selected. As isknown in the art, the anodization current density required to formporous silicon 110, 210 may depend in part on the type of siliconsubstrate used, such as whether the substrate is lightly or heavilyp-type (boron doped) or n-type (phosphorus doped).

In other alternative embodiments of the invention, the nanocrystallineporous silicon substrate 110, 210 may be incorporated into a MEMS devicecomprising a variety of detectors 420, sensors, electrodes, otherelectrical components, mechanical actuators, etc. using known chipmanufacturing techniques. In certain embodiments, such manufacturingprocedures may occur before and/or after formation of the porous siliconsubstrate 110, 210 and/or coating with a Raman sensitive metal 150.

Example 2 Metal Coating of Porous Silicon by Thermal Decomposition

FIG. 1 illustrates an exemplary method for uniformly impregnating metal150 into nanoporous silicon 110. The surface of the porous silicon 110is oxidized to silicon dioxide 120 (FIG. 1B). A metal salt solution 130is diffused into the porous matrix 110 (FIG. 1C) and dried (FIG. 1E).The dried metal salt 140 is thermally decomposed inside the pores toform a uniform metal layer 150 (FIG. 1F). Oxidation of the poroussilicon surface 110 enables complete wetting of porous silicon 110 inthe metal salt solution 130, while preventing spontaneous immersioncoating, which causes pore blockage. The dry metal salt 140 is thermallydecomposed in a furnace and pure metal 150 is deposited on the sidewalls of the nanopores. A uniform, thin metal 150 coating of nanoporoussilicon 110 may be obtained without plugging the pores, as oftenobserved with standard methods of metal 150 infiltration into nanoporoussilicon 110. Currently available plating methods are also diffusionlimited, resulting in non-uniform metal 150 deposition that can decreasethe reproducibility of analyte detection, depending upon where in themetal-coated substrate 110 the analyte is located.

An optimal immersion time and high metal ion concentration are needed tomake the metal 150 coat the entire porous structure 110. Theserequirements can be satisfied by oxidizing the surface of porous silicon110, either by chemical oxidation or plasma oxidation, prior to exposureto a metal salt solution 130 (FIG. 1B). Oxidation prevents spontaneousimmersion plating by stabilizing the porous surface 110. The oxidizedporous silion 110 may thus be immersed in highly concentrated metal saltsolution 130 without causing pore blockage (FIG. 1C). Excessive metalsalt solution 130 may be removed, for example by blowing nitrogen gas(FIG. 1D). The solvent is evaporated to increase absorption of metalsalt 140 on the porous surface 110 (FIG. 1E). The metal salts 140 arethermally decomposed (FIG. 1F) to form a uniform deposit of Raman activemetal 150 on the surface of the porous silicon substrate 110.

In a non-limiting example a porous silicon substrate 110 was formed byelectrochemical etching in a 15% HF solution, exposing boron dopedcrystalline silicon to a current density of 50 mA/cm². The poroussilicon substrate 110 was subjected to plasma oxidation in a Technicsoxygen plasma chamber at an oxygen flow rate of 50 sccm (standard cubiccentimeters) and radiofrequency power of 300 W (watts) for 20 min,resulting in formation of an approximately 50 Å (Angstrom) silicondioxide 120 layer on the surface of the pores. Alternatively, chemicaloxidation in piranha solution may be used (e.g.,http://www-device.eecs.Berkeley.edu/˜daewon/labweek7.pdf). The silicondioxide 120 layer passivates the silicon dangling bond, preventing fastimmersion coating.

The oxidized porous silicon 110 was dipped in a 1 M AgNO₃ solution 130for 20 min at room temperature to completely wet the pores with silvernitrate solution 130. Excessive silver nitrate solution 130 was removedby nitrogen gun drying to prevent pore closure by excessive silver 150deposition. The solvent was removed from the remaining silver nitratesolution 130 by drying at 100° C. for 20 min. At this stage all thesolvent was evaporated and dry silver nitrate salt 140 was absorbed onthe surface of pores, resulting in an observable brown color on thesurface of the porous silicon 110.

Thermal decomposition was performed for 30 min at 500° C. in an ambientpressure furnace, resulting in the decomposition of the dry silvernitrate salt 140 to silver metal 150. The method disclosed hereinresulted in the formation of a highly uniform deposit of silver metal150 on the surface of the porous silicon substrate 110, as shown in FIG.5. FIG. 5 illustrates the silver depth profile obtained on nanoporoussilicon 110, as determined by Rutherford backscattering spectroscopyanalysis. The silver depth profile was compared for nanoporous silicon110 treated by conventional diffusion limited immersion plating in a 1mM AgNO₃ solution 130 for 2.5 min (FIG. 5A) versus the method of thepresent Example (FIG. 5B). As can be seen, the present method resultedin a highly uniform silver 150 deposit, of much greater penetrationdepth compared to the standard method (FIG. 5A and FIG. 5B). The presentmethod resulted in a uniform silver 150 deposit up to about 10 μm indepth (FIG. 5B), while the standard method resulted in a highlynon-uniform deposit of less than 3 μm in depth (FIG. 5A). The Rutherfordbackscattering data were corrected using scanning electron microscopyanalysis to determine the actual thickness of the porous silicon 110layer.

Comparing the distribution of silver 150 versus silicon using thepresent method (FIG. 5B), it is observed that the silver 150 depositionis uniform down to the level at which the silicon density reaches amaximum. That is, the data of FIG. 5B indicate that the metal deposit150 obtained with the present method extends homogeneously all the wayto the bottom of the pores in the porous silicon substrate 110. It isclear that using the standard method (FIG. 5A) the metal deposit 150ends well before the bottom of the pores.

Example 3 Raman Detection of Analytes

A Raman active metal-coated substrate 110, 210 formed as disclosed abovemay be incorporated into a system 400 for Raman detection,identification and/or quantification of analytes, as exemplified in FIG.4. The substrate 110, 210 may be incorporated into, for example, a flowthrough cell, connected via inlet and outlet channels to one or moresolvent reservoirs 220 and a waste reservoir 230. Alternatively, theinlet channel may be connected to one or more other devices, such as asample injector and/or reaction chamber. Analytes may enter the flowthrough cell and pass across the Raman active substrate 110, 210, wherethey may be detected by a Raman detection unit. The detection unit maycomprise a Raman detector 420 and a light source 410, such as a laser.The laser 410 may emit an excitation beam, activating the analytes andresulting in emission of Raman signals. The Raman signals are detectedby the detector 420. In certain embodiments of the invention, thedetector 420 may be operably coupled to a computer that can process,analyze, store and/or transmit data on analytes present in the sample.

In an exemplary embodiment of the invention, the excitation beam isgenerated by a titanium:sapphire laser 410 (Tsunami by Spectra-Physics)at a near-infrared wavelength (750˜950 nm) or a galium aluminum arsenidediode laser 410 (PI-ECL series by Process Instruments) at 785 nm or 830nm. Pulsed laser beams or continuous beams may be used. The excitationbeam is reflected by a dichroic mirror (holographic notch filter byKaiser Optical or an interference filter by Chroma or Omega Optical)into a collinear geometry with the collected beam. The reflected beampasses through a microscope objective (Nikon LU series), and is focusedonto the Raman active substrate 110, 210 where target analytes arelocated. The Raman scattered light from the analytes is collected by thesame microscope objective, and passes the dichroic mirror to the Ramandetector 420. The Raman detector 420 comprises a focusing lens, aspectrograph, and an array detector. The focusing lens focuses the Ramanscattered light through the entrance slit of the spectrograph. Thespectrograph (RoperScientific) comprises a grating that disperses thelight by its wavelength. The dispersed light is imaged onto an arraydetector (back-illuminated deep-depletion CCD camera byRoperScientific). The array detector is connected to a controllercircuit, which is connected to a computer for data transfer and controlof the detector function.

In various embodiments of the invention, the detection unit is capableof detecting, identifying and/or quantifying a wide variety of analyteswith high sensitivity, down to single molecule detection and/oridentification. In certain embodiments of the invention, the analytesmay comprise single nucleotides that may or may not be Raman labeled. Inother embodiments, one or more oligonucleotide probes may or may not belabeled with distinguishable Raman labels and allowed to hybridize totarget nucleic acids in a sample. The presence of a target nucleic acidmay be indicated by hybridization with a complementary oligonucleotideprobe and Raman detection using the system 400 of FIG. 4. Alternatively,amino acids, peptides and/or proteins of interest may be detected and/oridentified using the disclosed methods and apparatus. The skilledartisan will realize that the methods and apparatus are not limiting asto the type of analytes that may be detected, identified and/orquantified, but rather that any analyte, whether labeled or unlabeled,that can be detected by Raman detection may be analyzed within the scopeof the claimed subject matter.

Example 4 Detection of Rhodamine 6G (R6G) by SERS

FIG. 6 illustrates the use of the disclosed methods, systems 400 andapparatus for detection and identification of an exemplary analyte,rhodamine 6G (R6G) dye molecules. R6G is a well-characterized dyemolecule that may be obtained from standard commercial sources, such asMolecular Probes (Eugene, Oreg.). A 114 μM (micromolar) solution of R6Gwas prepared and analyzed by surface enhanced Raman spectroscopy (SERS),using a plasma-oxidized, dip and decomposed (PODD) silver-coated poroussilicon substrate 110 that was prepared by the method of Examples 1 and2. Porous silicon substrates 110 of varying degrees of average porositywere prepared by varying the etching conditions. The R6G solution wasdiffused into the PODD silver-coated substrate 110 and analyzed by SERS,according to the method of Example 3, using an excitation wavelength of785 nm. A chemical enhancer (lithium chloride or sodium bromide, about 1μM concentration) was added to enhance the Raman signal.

The resulting SERS emission spectra, obtained in PODD silver-coatedporous substrates 110 of varying porosity, are shown in FIG. 6. FIG. 6shows SERS emission spectra for 114 μM R6G obtained at averageporosities, in order from the lowest trace to the highest trace, of 52%,55%, 65%, 70% and 77%. As indicated in FIG. 6, the intensity of the SERSemission peaks increases with increasing average porosity in this range,with a highest intensity observed at 77% average porosity. Increasingthe porosity above 77% pushes the porous silicon layer 110 into anon-stable materials regime, which can result in physical separation ofthe porous layer 110 from the bulk silicon substrate. At 77% porosity,scanning electron micrographs showed pore diameters of about 32 nm inwidth (not shown).

At 77% average porosity, a seven order of magnitude (10⁷) increase inintensity of the Raman emission spectrum was observed. This compareswith an approximately six order of magnitude enhancement observed on aroughened silver 150 plate (not shown). Although the intensity of theSERS emission peaks increased as a function of average porosity, thewavelengths of the emission peaks did not vary (FIG. 6), allowing theidentification of R6G independent of the average porosity used. With anestimated detection volume of 1.25×10⁻¹⁶ liters, the correspondingnumber of molecules of rhodamine 6 G detected was approximately 9molecules.

Additional studies were performed with a solution of adenine, which is amore biologically relevant target molecule. Unique spectroscopicfeatures were detected from a 90 μM solution of adenine on a poroussilicon substrate 110 coated with silver 150.

All of the METHODS, SYSTEMS 400 and APPARATUS disclosed and claimedherein can be made and used without undue experimentation in light ofthe present disclosure. It will be apparent to those of skill in the artthat variations may be applied to the METHODS, SYSTEMS 400 and APPARATUSdescribed herein without departing from the concept, spirit and scope ofthe claimed subject matter. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the claimed subject matter.

1. A method comprising: a) obtaining a porous silicon substrate andinserting the porous silicon substrate in a microfluidic channel; b)oxidizing the surface-of the substrate to form silicon dioxide; c)wetting the substrate with a metal salt solution; and d) thermallydecomposing the metal salt to form a metal deposit on the oxidizedsurface of the substrate.
 2. The method of claim 1, further comprisingremoving excess metal salt solution from the substrate.
 3. The method ofclaim 2, further comprising removing the solvent from the remainingmetal salt solution.
 4. The method of claim 3, wherein the solvent isremoved by drying.
 5. The method of claim 1, wherein the metal depositcomprises at least one Raman active metal selected from the groupconsisting of silver, gold, platinum, copper and aluminum.
 6. The methodof claim 1, wherein the metal salt solution comprises silver nitrate. 7.The method of claim 6, wherein the solution comprises between 0.5 molarand 5.0 molar silver nitrate.
 8. The method of claim 1, wherein theporous silicon substrate has an average porosity of about 10%, 15%, 20%,25%, 30%, 35% 40%, 50%, 55%, 60%, 65% 70%, 75% or 80%.
 9. The method ofclaim 1, wherein the porous silicon substrate is oxidized by plasmaoxidation, chemical oxidation or thermal annealing.
 10. The method ofclaim 1, wherein the metal salt is thermally decomposed by heating to atemperature above 573 K.